Phage therapy of E coli infections

ABSTRACT

The present invention relates to bacteriophage therapy. More particularly, the present invention relates to novel bacteriophages having a high specificity against Escherichia coli strains, their manufacture, components thereof, compositions comprising the same and the uses thereof in phage therapy.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP2015/050355, filed Jan. 9, 2015.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jun. 24, 2016 and is 1552 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to novel bacteriophages, compositionscomprising the same, their manufacture, and the uses thereof. Theinvention is particularly adapted for the treatment of an infection in amammal and for improving a subject condition by modifying the flora insaid subject.

BACKGROUND OF THE INVENTION

Bacteriophages (or phages) are small viruses displaying the ability toinfect and kill bacteria while they do not affect cells from otherorganisms. Initially described almost a century ago by William Twort,and independently discovered shortly thereafter by Felix d'Herelle, morethan 6000 different bacteriophages have been discovered and describedmorphologically, including bacterial and archeal viruses. The vastmajority of these viruses are tailed while a small proportion arepolyhedral, filamentous or pleomorphic. They may be classified accordingto their morphology, their genetic content (DNA vs. RNA), their specifichost, the place where they live (marine virus vs. other habitats), andtheir life cycle. As intra-cellular parasites of bacterial cells, phagesdisplay different life cycles within the bacterial host: lytic,lysogenic, pseudo-lysogenic, and chronic infection (Weinbauer, 2004;Drulis-Kawa, 2012). Lytic phages cause lysis of the host bacterial cellas a normal part of their life cycles. Lysogenic phages (also termedtemperate phages) can either replicate by means of the lytic life cycleand cause lysis of the host bacterium, or they can incorporate their DNAinto the host bacterial DNA and become noninfectious prophages. Whateverthe type of cycle of a phage life, the first step is the attachment toreceptors of the bacterial cell wall before phages may enter thebacteria. This specific process influences the spectrum of the possiblephage-bacteria interactions.

Bacteriophages are commonly used as research tools to modify bacteria inlaboratory experiments.

Because of their target host cell specificity, the use of phages as atherapy to treat acute and chronic infections has been considered,particularly in dermatology, ophthalmology, urology, stomatology,pediatrics, otolaryngology or surgery. This concept of therapeutic useof phages to treat bacterial infection was, however, highlycontroversial from the very beginning and not widely accepted by thepublic or medical community. Early studies were widely criticized forlack of appropriate controls and inconsistent results. The lack ofreproducibility and many conflicting results obtained in the variouspublished studies led the Council on Pharmacy and Chemistry of theAmerican Medical Association to conclude that the evidence for thetherapeutic value of lytic filtrates was for the most partcontradictory, unconvincing, and recommended additional research toconfirm its purported benefits.

Since the introduction of antibiotics in the 1940s, little attention waspaid to this field of therapeutics, especially in the Western world. Butthe extensive use of antibiotics has led to the widespread emergence andspread of antibiotic-resistant bacteria around the world, causingincreasingly serious problems. It has therefore become a majortherapeutic challenge to overcome the limited therapeutic optionsremaining to treat major multi-drug resistant microbes.

In addition, many pathogenic microorganisms reside within bio films,which bio films cause additional problems when designing newanti-microbial agents. In this regard, bacteria growing as a biofilmrather than in single-celled (“planktonic”) forms tend to beparticularly resistant to anti-microbial agents and to be particularlydifficult for the host immune system to render an appropriate response.

E. coli, a Gram-negative, short rod-shaped bacterium belonging to thegenus Escherichia and the family Enterobacteriaceae, shows highdiversity and frequency in human or animal microbial flora. It wasrevealed that while most strains of E. coli are non-pathogenic, they cancause opportunistic infections. Furthermore, some E. coli strains arehighly pathogenic and can cause diverse diseases and sepsis in mammals,including humans. Several reports associate the enterobacteriumEscherichia coli with skin and soft tissue infections (SSTIs), one ofthe most common infections in patients of all age groups. In somemoderate or severe cases, these infections require hospitalization andparenteral therapy. E. coli was notably found to be the causative agentof neonatal omphalitis (Fraser and al, 2006), cellulitis localized tolower or upper limbs (Brzozowski and al, 1997, Corredoira and al, 1994),necrotizing fasciitis (Afifi and al, 2008; Krebs and al, 2001), surgicalsite infections (Tourmousoglou and al, 2008), infections after burninjuries (Rodgers and al, 2000), and others. A study monitoring SSTIsduring a 7-year period and encompassing three continents (Europe, LatinAmerica, and North America) showed E. coli to be an important causativeagent, since it was the third-most prevalent isolated species. E. colitherefore deserve specific and targeting therapy, especially taking intoaccount the dramatic decline in antibiotic susceptibility of pathogenicE. coli strains in recent years, their diversity, and their substantialpresence in microbial flora.

Furthermore, E. coli bacteria are able to form biofilms, contributing totheir increased resistance to antibiotics. Such biofilms may comprisemore than one type of bacteria supported and surrounded by an excretedextracellular matrix and assist bacteria to colonize various surfaces.Bio films allow bacteria to attach to surfaces and to attain populationdensities which would otherwise be unsupportable, imparting increasedresistance to not only antibiotics but many environmental stressesincluding toxins such as heavy metals, bleaches and other cleaningagents. It is known that bacteria within biofilms can be 100 to 1000times more resistant to antibiotics than the same strain of bacteriagrowing in planktonic forms. Such an increased resistance means thatbacteria that are apparently sensitive to antibiotics in a laboratorytest may be resistant to therapy in a clinical setting. Even if some arecleared, bio films may provide resistant reservoirs permitting rapidcolonization once antibiotics are no longer present. It is thereforeobvious that biofilms are major factors in many human diseases. Chemicaltreatments are unsuited to use against bio films since this is preciselywhat they have evolved to counter. Physical abrasion does provide a meanto disrupt biofilms. Unfortunately, many surfaces where bio filmssupports bacterial pathogenesis are poorly suited to rigorous abrasion,i.e. bones, joints, implanted medical devices, etc. For example, thesurfaces of wounds or burns are extremely sensitive and delicate. Evenwhere abrasion is both suitable and in routine use, clearing of biofilmsis limited. Oral plaque on the surface of teeth is a biofilm and ispartially cleared by regular brushing. However, bacteria are maintainedon unbrushed surfaces (for example in the gaps between teeth) and canrecolonize cleared surfaces both rapidly and effectively. From this, itis clear that existing approaches to clearing bio films are of limitedefficacy.

The capability for quick adaptation and their ability to form bio filmsare the main reasons that identify E. coli as opportunistic pathogens.They have acquired the status of hospital pathogens, and may be isolatedfrom clinical samples taken from the wounds, sputum, bladder, urethra,vagina, ears, eyes and respiratory tract. The emergence of resistance tothe most powerful new antibiotics in such clinical E. coli strains,occurring even during treatment, makes the fight with E. coli hospitalpathogens a great problem.

Furthermore, it has been reported that the pathological or physiologicalcondition of a subject is influenced by the balance of microorganisms inthe flora of the subject. Accordingly, modifying the microbial flora, ormodifying said balance, or restoring said balance, by destroying E. colipopulation, is also a valuable approach for improving a subjectcondition.

Therefore, there is a great need for new antibacterial agents orcompositions that can be used to destroy E. coli strains, even whenorganized in bacterial biofilms, suitable for use in human or animaltherapy as well as for decontaminating materials.

SUMMARY OF THE INVENTION

The inventors have isolated and characterized new bacteriophagespresenting specific lytic activity to Escherichia coli (E. coli), andwhich can be used as active agents in pharmaceutical or veterinarypreparations, particularly to treat E. coli bacterial infections or tomodify microbial balance in a subject. The new bacteriophages of theinvention exhibit strong lytic activity, high selectivity, and can bycombined to induce controlled destruction of a very large spectrum of E.coli cells.

An object of the invention is to provide antibacterial compositionscomprising at least one, preferably at least two bacteriophages havinglytic activity against an Escherichia coli strain, said bacteriophagesbeing selected from the bacteriophages having a genome comprising anucleotide sequence of anyone of SEQ ID NOs: 1 to 15 or a sequencehaving at least 90% identity thereto.

A further object of the present invention concerns a bacteriophagehaving lytic activity to an Escherichia coli strain, said bacteriophagehaving a genome comprising a nucleotide sequence selected from anyone ofSEQ ID NOs: 1 to 15 or a sequence having at least 90% identity thereto,preferably at least 97% identity thereto. In a specific embodiment, thebacteriophages of the invention exhibit lytic activity to multi drugresistant strains, in particular to an antibiotic resistant pathogenicE. coli, such as, preferably Extended-Spectrum Beta-Lactamases (ESBL)strains or verotoxin-producing E. coli (VTEC) strains.

Another object of the invention relates to a bacteriophage which isselected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002,BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, or BP1229, having agenome comprising the nucleotide sequence of SEQ ID NOs: 1 to 15,respectively, and variants thereof, wherein said variants retain aphenotypic characteristic of said bacteriophage, and wherein saidbacteriophage and variants thereof have lytic activity against an E.coli strain.

Another object of the invention resides in a composition comprising atleast one bacteriophage as defined above. In a particular embodiment,the compositions of the invention comprise at least two distinctbacteriophages as defined above, preferably at least three, even morepreferably at least four distinct bacteriophages as defined above. Aspecific composition of the invention comprises a combination of all ofbacteriophages BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002,BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and BP1229.

In another aspect, the invention is related to a bacteriophage havinglytic activity to a pathogenic E. coli strain, wherein the bacteriophageis specific for E. coli, active against antibiotic-resistant E. colistrains, and has a productive lytic effect below 15.

The invention further concerns an isolated nucleic acid sequencecontained in a bacteriophage of the invention, and an isolatedpolypeptide encoded by said isolated nucleic acid.

Another object of the invention is a composition comprising apolypeptide as defined above.

A further object of the invention is a composition comprising a nucleicacid as defined above.

The compositions of the invention typically further comprise apharmaceutically or veterinary acceptable excipient or carrier. They maybe liquid, semi-liquid, solid or lyophilized.

Another object of the invention relates to a bacteriophage, nucleicacid, polypeptide or composition as defined above, for use in thetreatment of an infection in a mammal, for modifying the microbial florain a mammal, for decontaminating a material and/or for killing an E.coli bacterium or for compromising the integrity of a bacterial biofilm.

The invention relates also to the use of one or several lyticbacteriophages to improve a subject condition by modifying the microbialflora in said subject. The microbial flora may be modified bycorrecting, adapting or restoring a proper balance of microorganisms insaid flora.

The invention also relates to a method for treating an infection in amammal, comprising the administration to said mammal of at least onebacteriophage, nucleic acid, polypeptide or composition as definedabove.

The invention also relates to a method for treating a surface ormaterial suspected of being contaminated with an E. coli bacterium,comprising applying to said surface or material at least onebacteriophage, nucleic acid, polypeptide or composition as definedabove. The surface or material may be a surface of e.g., any device,vessel or laboratory material, cloth, etc.

Another object of the invention relates to a method for predicting ordetermining efficacy of a bacteriophage therapy in a subject, whereinthe method comprises the step of determining in vitro a lytic activityof one or more bacteriophages of the invention to an E. coli strain froma sample of said subject, a lytic activity of one or more bacteriophagesof the invention to at least one E. coli strain from said sample beingindicative of an efficient treatment. The method further optionallycomprises the step of treating the subject with at least onebacteriophage having a lytic activity to an E. coli strain from a sampleof said subject.

In another aspect, the invention provides a method for selecting asubject or determining whether a subject is susceptible to benefit froma bacteriophage therapy, wherein the method comprises the step ofdetermining in vitro a lytic activity of one or more bacteriophages ofthe invention to an E. coli strain from a sample of said subject, alytic activity of one or more said bacteriophages of the invention to atleast one E. coli strain being indicative of a responder subject.

The invention may be used in any mammal, preferably in human beings, orto treat any material, including laboratory materials or medicaldevices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: In vitro efficiency of Bacteriophages of the invention oncombinations of E. coli strains at various MOIs.

FIG. 2: In vivo efficacy of Bacteriophages of the invention oncombinations of E. coli strains at various dosages.

FIG. 3: Efficacy of bacteriophages of the invention on SH113 E. colistrain-mediated infection in vivo. Φ IV: intravenous treatment; Φ IP:intraperitoneal treatment; Φ SC: subcutaneous treatment; Temp Inf.:infected untreated control; Tem Genta.: infected gentamicin-treatedcontrol.

FIG. 4: Efficacy of bacteriophages of the invention on SH113 E. colistrain-mediated infection in vivo: dose effect. Φ conc.: fullconcentration (10⁸ pfu/ml); Φ 1/10: 10 fold diluted concentration; Φ1/100:100 fold diluted concentration; Φ 1/1000: 1000 fold dilutedconcentration; Tem infect.: infected antibiotic treated control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel bacteriophages, componentsthereof, compositions comprising the same, their manufacture, and theuses thereof as antibacterial agents, particularly for the treatment ofan infection in a mammal and for improving a subject condition bymodifying the microbial flora in said subject.

Definitions

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “bacteriophage” or “phage” refers to afunctional phage particle comprising a nucleic acid genome packaged in aproteinaceous envelope or capsid. The term also refers to portions ofthe bacteriophage, including, e.g., a head portion, or an assembly ofphage components, which provide substantially the same functionalactivity.

The term “phenotypic characteristic” designates more preferably themorphology and/or host-range of a bacteriophage. Methods for phenotypingbacteriophages are well known per se in the part and include, forexample, determining bacterial host range and/or activity against thebiofilm produced by certain bacterial strains.

The term “lytic activity” as used in the invention designates theproperty of a bacteriophage to cause lysis of a bacterial cell. Thelytic activity of a bacteriophage can be tested on E. coli strainsaccording to techniques known per se in the art (see also experimentalsection).

The term “variant” of a reference bacteriophage designatesbacteriophages having variation(s) in the genomic sequence and/orpolypeptide(s) encoded thereby as compared to said referencebacteriophage, while retaining the same phenotypic characteristic as thereference bacteriophage. Variants typically comprise e.g., silentmutations, conservative mutations, minor deletions, and/or minorreplications of genetic material, and retain phenotypic characteristicsof the reference bacteriophage. In a preferred embodiment, the variantof the invention retain any observable characteristic or property thatis dependent upon the genome of the bacteriophage of the invention, i.e.phenotypic characteristics of said bacteriophage and/or lytic activityagainst the E. coli strains. Preferred variants have less than 5%nucleic acid variation as compared to the genome of the referencebacteriophage, even more preferably less than 4%, more preferably lessthan 2%. Alternatively, or in combination, variants have preferably lessthan 5% amino acid variation in a coded polypeptide sequence as comparedto a polypeptide of the reference bacteriophage.

The term “% identity” in relation to nucleic acid sequences designatesthe level of identity or homology between said sequences and may bedetermined by techniques known per se in the art. Typically, the %identity between two nucleic acid sequences is determined by means ofcomputer programs such as GAP provided in the GCG program package(Program Manual for the Wisconsin Package, Version 8, August 1996,Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711)(Needleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48, 443-453). With settings adjusted to e.g., DNA sequences(particularly: GAP creation penalty of 5.0 and GAP extension penalty of0.3), nucleic acid molecules may be aligned to each other using thePileup alignment software available as part of the GCG program package.

The term “fragment” of a nucleic acid designates typically a fragmenthaving at least 10 consecutive nucleotides of said nucleic acid, morepreferably at least 15, 20, 25, 30, 35, 40, 50 or more consecutivenucleotides of said nucleic acid.

The term “fragment” of a polypeptide designates typically a fragmenthaving at least 5 consecutive amino acids of said polypeptide, morepreferably at least 10, 15, 20, 30, 40, 50 or more consecutive aminoacids of said polypeptide.

The term “ESBL E. coli strain” refers to antibiotic-resistant E. coli,more specifically to Extended-Spectrum Beta-Lactamases-producing E. colistrains.

The term “VTEC” refers to another type of antibiotic-resistant E. colistrains, more specifically to verotoxin-producing E. coli strains.

The term “specific” or “specificity” in relation to a bacteriophagerefers to the type of host that said bacteriophage is able to infect.Specificity is usually mediated by the tail fibers of bacteriophages,that are involved in the interaction with receptors expressed on cells.A bacteriophage “specific” for E. coli more preferably designates abacteriophage which can infect one or several E. coli strains and whichcannot infect non-E. coli bacteria under physiological conditions.

As used herein, the term “polypeptide” refers to polypeptides of anysize, including small peptides of e.g., from 5 to 20 amino acids, longerpolypeptides, proteins or fragments thereof.

The term “PLE” or “Productive Lytic Effect” designates the ratio betweenburst size and productive lytic time of a given bacteriophage. Burstsize and productive lytic time are parameters defining phage-hostinteraction and correspond, respectively, to the mean yield ofbacteriophage particles produced by infection of one bacterium by onephage, and to the time taken by a free bacteriophage to lyse a bacterialcell.

In the context of the present specification, the term “isolatedbacteriophage” should be considered to mean material removed from itsoriginal environment in which it naturally occurs. In relation to abacteriophage, the term designates, particularly a phage that is e.g.,cultivated, purified and/or cultured separately from the environment inwhich it is naturally located. In relation to a nucleic acid orpolypeptide, the term “isolated” designates e.g., a nucleic acidmolecule or polypeptide which is separated from at least some of thecomponents of its natural environment such as, e.g., a protein, lipid,and/or nucleic acid.

The terms “pharmaceutically or veterinary acceptable” as used hereinrefers to any material (e.g., carrier, excipient or vehicle) that iscompatible for use in a mammalian subject. Such includes physiologicallyacceptable solutions or vehicles that are harmless or do not cause anysignificant specific or non-specific immune reaction to an organism ordo not abrogate the biological activity of the active compound. Forformulation of the composition into a liquid preparation, saline,sterile water, Ringer's solution, buffered physiological saline, albumininfusion solution, dextrose solution, maltodextrin solution, glycerol,ethanol, and mixtures thereof may be used as a pharmaceutically orveterinary acceptable excipient or carrier. If necessary, otherconventional additives such as thickeners, diluents, buffers,preservatives, surface active agents, antioxidants and bacteriostaticagents may be added. Further, diluents, dispersants, surfactants,binders and lubricants may be additionally added to the composition toprepare injectable formulations such as aqueous solutions, suspensions,and emulsions, oral formulations such as pills, capsules, granules, ortablets, or powdered formulations.

As used herein, “PFU” means plaque forming unit, as it is well definedin the art. Lytic bacteriophages lyse the host cell, causing a zone ofclearing (or plaque) on a culture plate. Theoretically, each plaque isformed by one phage and the number of plaques multiplied by the dilutionfactor is equal to the total number of phages in a test preparation.

The term “treatment” or “therapy” designates both a curative treatmentand/or a prophylactic treatment of a disease. A curative treatment isdefined as a treatment resulting in cure or a treatment alleviating,improving and/or eliminating, reducing and/or stabilizing the symptomsof a disease or the suffering that it causes directly or indirectly. Aprophylactic treatment comprises both a treatment resulting in theprevention of a disease and a treatment reducing and/or delaying theincidence of a disease or the risk of its occurrence.

The term “mammal” includes human subjects as well as non-human mammalssuch as pets (e.g., dogs, cats), horses, ruminants, sheep, goats, pigs,etc.

The term “biofilm” as used herein designates to be a heterogeneousbacterial formation growing on various surfaces; preferably a bacterialcommunity growing embedded in an exopolysaccharide matrix adhered ontosolid biological or non-biological surfaces.

The term “compromise” as used herein refers to any alteration of theintegrity. By compromising a bacterial bio film, it is understood apenetration of the bio film by bacteriophage, an infection of biofilm-associated bacteria and/or a lysis thereof and/or a partial or anentire clearing of the biofilm (i.e. by stopping colonization and/ordisrupting bio films).

The term “sample”, as used herein, means any sample containing cells.Examples of such samples include fluids such as blood, plasma, saliva,or urine as well as biopsies, organs, tissues or cell samples. Thesample may be treated prior to its use.

As used herein, the term “subject” or “patient” refers to an animal,preferably to a mammal, even more preferably to a human, including adultand child. However, the term “subject” also encompasses non-humananimals, in particular mammals such as dogs, cats, horses, cows, pigs,sheep and non-human primates, among others.

The term “efficacy” of treatment or “response” to a bacteriophagetherapy as used herein refers to a treatment which results in a decreasein the number of E. coli strains in a subject after bacteriophagetreatment when compared to the number of E. coli strains beforetreatment. A “good responder” subject refers to a subject who shows orwill show a clinically significant recovery when treated with abacteriophage therapy.

The term “Cocktail” or composition of bacteriophages designates acombination of two or more different bacteriophages. The bacteriophagesin a cocktail/composition are preferably formulated together, i.e., in asame vessel or packaging, although they may be used as kits of partswherein the (or some of the) bacteriophages are formulated or packagedseparately and combined when used or administered.

DESCRIPTION OF EMBODIMENTS

The present invention is related to novel bacteriophage therapies. Moreparticularly, the present invention relates to novel bacteriophageshaving a high specificity against Escherichia coli strains, theirmanufacture, components thereof, compositions comprising the same andthe uses thereof in phage therapy.

Bacteriophages:

In a first aspect, the invention discloses the isolation andcharacterization of novel bacteriophages that are specific for E. colistrains and present, either alone or in combination(s), remarkable hostrange spectrum of lytic activity. These bacteriophages have beenselected from environmental samples, isolated, and characterized. Asindicated, the bacteriophages are, individually and in combination(s),active against E. coli strains. They are remarkable effective againstpathogenic E. coli strains, such as antibiotic-resistant E. colistrains. Furthermore, bacteriophages of the invention have a remarkableproductive lytic effect (“PLE”) below 15, more preferably below 10 andstill more preferably between 0.1 and 10. Furthermore, thebacteriophages of the invention are specific for E. coli strains, i.e.,they do not causes lysis of non-E. coli bacteria. As will be illustratedfurther, the invention shows that these bacteriophages can be combinedand formulated in conditions suitable for use as pharmaceutical orveterinary agents to exhibit targeted and very potent antibacterialeffect against a controlled spectrum of E. coli strains.

More specifically, the following bacteriophages have been selected andcharacterized. Their corresponding nucleic acid sequences are alsoindicated.

TABLE 1 SEQ ID number Bacteriophage SEQ ID NO: 1 BP539 SEQ ID NO: 2BP700 SEQ ID NO: 3 BP753 SEQ ID NO: 4 BP814 SEQ ID NO: 5 BP953 SEQ IDNO: 6 BP954 SEQ ID NO: 7 BP970 SEQ ID NO: 8 BP1002 SEQ ID NO: 9 BP1151SEQ ID NO: 10 BP1155 SEQ ID NO: 11 BP1168 SEQ ID NO: 12 BP1176 SEQ IDNO: 13 BP1197 SEQ ID NO: 14 BP1226 SEQ ID NO: 15 BP1229

The lytic profile of these bacteriophages has been determined on a broadnumber of E. coli strains. These bacteriophages have been selected fortheir potency and combination potential, as disclosed in the followingtable. In this table, the lytic effect of the bacteriophages onreference and pathogen-resistant strains are presented, to confirm thehigh lytic potential.

TABLE 2 Phage EC 539 700 753 814 953 954 970 1002 1151 1155 1168 11761197 1226 1229 15 phages  1 K12/DH5 + + +* + + + + +* + +* +  2ECOR1 + + +  3 ECOR2 + +/− + + +/− +/− + +  4 ECOR4 + + +  5 ECOR5 + + + 6 ECOR10 + +/− +  7 ECOR13 + + + +/− +/− + + + +/− +  8 ECOR15 + ++/− + + + + + + +  9 ECOR24 + +/− + + + 10 ECOR28 + + + + +/− + +/−+/− + 11 ECOR35 + +* + +/− + +/− + 12 ECOR38 + + + +/− + 13 ECOR40 + + ++/− + 14 ECOR46 + + + + + + + + + 15 ECOR48 + +/− + + +/− + + 16ECOR50 + + +/− +/− +/− +/− +/− + 17 ECOR54 +* + + + + + + + 18 ECOR55 +*+* +* + + + + + + 19 ECOR56 +* + +* +* +/− + 20 ECOR59 + + +/− +* +*+/− + +* + + 21 ECOR60 + + + +/− +* + +/− + 22 ECOR62 + + + + 23ECOR64 + + + + + + 24 ECOR71 + +/− + +/− + + 25 ECOR72 + + +/− +/− +/−+/− +

Boxes with an asterisk (“*”) are examples of producing E. coli strains.

As can be seen from table 2, the phages have individually very stronglytic power, and combinations (or cocktails) of these bacteriophages maybe produced that are able to kill all of the tested E. coli strains,thereby producing broad spectrum antibacterial compositions.

As an illustration, a cocktail of all 15 phages of the invention is ableto effectively kill all bacteria listed in Table 2.

Moreover, the specificity of the bacteriophages has been tested on manynon-E. coli strains. More particularly, the experimental sectiondemonstrates that the bacteriophages of the invention have no lyticeffect on bacteria selected from Pseudomonas aeruginosa, Acinebacterbaumanii, Enterobacter aerogenes, Enterobacter ashuriae, Enterobactercloacae, Klebsiella pneumonia, Porteus mirabilis, Staphylococus aureus,Stenotrophomonas maltophila and/or Serratia marcescens.

A particular object of the invention thus resides in a bacteriophagehaving lytic activity to an E. coli strain and having a genomecomprising a nucleotide sequence selected from anyone of SEQ ID NOs: 1to 15 or a sequence having at least 97% identity thereto, preferably atleast 98% or 99% identity thereto.

A particular object of the invention thus resides in a bacteriophagehaving lytic activity to an E. coli strain and having a genome having orconsisting of a nucleotide sequence selected from anyone of SEQ ID NOs:1 to 15.

The bacteriophages of the invention can be prepared by standard culture,isolation and purification methods. For example, E. coli producingbacteria are cultured, infected by a sample of a bacteriophage, and thentreated to remove bacterial cells and debris. The enriched bacteriophagesolution can be plated in a medium, for example agar medium, withembedded susceptible host strains of E. coli to obtain plaques. Then,single plaque can be picked out for subsequent bacteriophagepurification and amplification. One or more cycles of selectiveamplification of bacteriophages of the invention may be performed, forexample by mixing bacteriophages with the competent E. coli, followed byaddition of a growth medium and incubation at selected test growingconditions. Following centrifugation, the cleared amplified supernatantis filtered through filter and subjected to another cycle of selectiveamplification or tested for presence of lytic activity. The titer ofphage in a suspension and the visualization of plaque morphology ofbacteriophages of the invention can be estimated by known methods, forexample by plaque counting. Additionally, processing bacteriophages ofthe invention in various form (liquid, lyophilized, etc.) for short-,long-, freeze- or any other kind of storage can be carried out by anysuitable method as it is well-known in the art (see Clark, 1962).

The activity of the bacteriophages of the invention can be assessed bymethods well-known in the art, such as plaque assay also known as doubleagar method, based on the growing of bacteriophage with potential hostbacteria and followed by assessing their ability to kill the hostbacterial cell. In the plaque assay method, the bacteriophage induceslysis of target E. coli strains after a period of incubation in softagar medium, resulting in zones of clearing on the plate known asplaques. In a preferred aspect, the bacteriophages of the inventionexhibit, either alone or in combination, lytic activity to a pathogenicE. coli strain, including to an antibiotic-resistant E. coli strain,such as an ESBL E. coli strain. Furthermore, variants of thesebacteriophages retaining a phenotypic (e.g., specificity and lyticactivity) of these bacteriophages can be produced and/or isolated bytechniques known per se in the art.

In a particular embodiment, the invention is related to BP539bacteriophage, or any variant thereof. BP539 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR54. BP539, or any variant thereof, is specific and has lyticactivity against K12/DH5, ECOR15, ECOR35, ECOR38, ECOR40, ECOR54,ECOR62, ECOR64 and/or ECOR71 strains. BP539 comprises a genomecomprising a sequence as set forth in SEQ ID NO: 1 or having at least80% identity, more preferably at least 85% identity, and still morepreferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ IDNO: 1. It is also provided an isolated nucleic acid sequence from BP539bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP539 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from a BP539bacteriophage of the invention. BP539 bacteriophage of the invention isalso characterized by a PLE below 15, more preferably below 10 and stillmore preferably of around 0.1.

In another particular embodiment, the invention is related to BP700bacteriophage, or any variant thereof. BP700 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR55. BP700, or any variant thereof, is specific and has lyticactivity against ECOR46, ECOR55, ECOR60 and/or ECOR64 strains. BP700comprises a genome comprising a sequence as set forth in SEQ ID NO: 2 orhaving at least 80% identity, more preferably at least 85% identity, andstill more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identityto SEQ ID NO: 2. It is also provided an isolated nucleic acid sequencefrom BP700 bacteriophage, or variant thereof. The invention alsoencompasses isolated polypeptides encoded by BP700 bacteriophage, orvariant thereof, or encoded by an isolated nucleic acid sequence fromBP700 bacteriophage of the invention.

BP700 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around2.

In still another aspect, the invention is related to BP753bacteriophage, or any variant thereof. BP753 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR56. BP753, or any variant thereof, is specific and has lyticactivity against ECOR10, ECOR48 and/or ECOR56 strains. BP753 comprises agenome comprising a sequence as set forth in SEQ ID NO: 3 or having atleast 80% identity, more preferably at least 85% identity, and stillmore preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 3. It is also provided an isolated nucleic acid sequence fromBP753 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP753 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP753bacteriophage of the invention.

BP753 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around2.

In another aspect, the invention is related to BP814 bacteriophage, orany variant thereof. BP814 bacteriophage, or any variant thereof, can beproduced or expanded in e.g., E. coli strain ECOR55. BP814, or anyvariant thereof, is specific and has lytic activity against ECOR46,ECOR50, ECOR55 and/or ECOR64 strains. BP814 comprises a genomecomprising a sequence as set forth in SEQ ID NO: 4 or having at least80% identity, more preferably at least 85% identity, and still morepreferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ IDNO: 4. It is also provided an isolated nucleic acid sequence from BP814bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP814 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP814bacteriophage of the invention.

BP814 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around0.3.

In another particular embodiment, the invention is related to BP953bacteriophage, or any variant thereof. BP953 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR55. BP953, or any variant thereof, is specific and has lyticactivity against K12/DH5, ECOR1, ECOR46, ECOR50, ECOR54, ECOR55, ECOR59and/or ECOR60 strains. BP953 comprises a genome comprising a sequence asset forth in SEQ ID NO: 5 or having at least 80% identity, morepreferably at least 85% identity, and still more preferably 90%, 92%,94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5. It is alsoprovided an isolated nucleic acid sequence from BP953 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP953 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP953 bacteriophage of theinvention. BP953 bacteriophage of the invention is also characterized bya PLE below 15, more preferably below 10 and still more preferably ofaround 3.

In another particular embodiment, the invention is related to BP954bacteriophage, or any variant thereof. BP954 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR35. BP954, or any variant thereof, is specific and has lyticactivity against ECOR24, ECOR35, ECOR38, ECOR40, ECOR46 and/or ECOR62strains. BP954 comprises a genome comprising a sequence as set forth inSEQ ID NO: 6 or having at least 80% identity, more preferably at least85% identity, and still more preferably 90%, 92%, 94%, 95%, 96%, 97%,98% or 99% identity to SEQ ID NO: 6. It is also provided an isolatednucleic acid sequence from BP954 bacteriophage, or variant thereof. Theinvention also encompasses isolated polypeptides encoded by BP954bacteriophage, or variant thereof, or encoded by an isolated nucleicacid sequence from BP954 bacteriophage of the invention.

BP954 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around3.

In still another aspect, the invention is related to BP970bacteriophage, or any variant thereof. BP970 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainK12/DH5. BP970, or any variant thereof, is specific and has lyticactivity against K12/DH5, ECOR1, ECOR2, ECOR5, ECOR10, ECOR13, ECOR15,ECOR28 and/or ECOR72 strains. BP970 comprises a genome comprising asequence as set forth in SEQ ID NO: 7 or having at least 80% identity,more preferably at least 85% identity, and still more preferably 90%,92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7. It is alsoprovided an isolated nucleic acid sequence from BP970 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP970 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP970 bacteriophage of theinvention.

BP970 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around5.

In another particular embodiment, the invention is related to BP1002bacteriophage, or any variant thereof. BP1002 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR56. BP1002, or any variant thereof, is specific and has lyticactivity against ECOR15, ECOR24, ECOR35, ECOR38, ECOR40, ECOR48, ECOR54,ECOR55, ECOR56, ECOR59, ECOR60 and/or ECOR64 strains. BP1002 comprises agenome comprising a sequence as set forth in SEQ ID NO: 8 or having atleast 80% identity, more preferably at least 85% identity, and stillmore preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 8. It is also provided an isolated nucleic acid sequence fromBP1002 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1002 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1002bacteriophage of the invention.

BP1002 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around5.

In another particular embodiment, the invention is related to BP1151bacteriophage, or any variant thereof. BP1151 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR56. BP1151, or any variant thereof, is specific and has lyticactivity against K12/DH5, ECOR2, ECOR13, ECOR15, ECOR24, ECOR35, ECOR38,ECOR40, ECOR46, ECOR48, ECOR50, ECOR54, ECOR56, ECOR59, ECOR60, ECOR62,ECOR64 and/or ECOR71 strains. BP1151 comprises a genome comprising asequence as set forth in SEQ ID NO: 9 or having at least 80% identity,more preferably at least 85% identity, and still more preferably 90%,92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9. It is alsoprovided an isolated nucleic acid sequence from BP1151 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP1151 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP1151 bacteriophage of theinvention.

BP1151 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around10.

In another aspect, the invention is related to BP1155 bacteriophage, orany variant thereof. BP1155 bacteriophage, or any variant thereof, canbe produced or expanded in e.g., E. coli strain ECOR59. BP1155, or anyvariant thereof, is specific and has lytic activity against K12/DH5,ECOR2, ECOR13, ECOR15, ECOR28, ECOR46, ECOR50, ECOR54, ECOR55, ECOR59and/or ECOR71 strains. BP1155 comprises a genome comprising a sequenceas set forth in SEQ ID NO: 10 or having at least 80% identity, morepreferably at least 85% identity, and still more preferably 90%, 92%,94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10. It is alsoprovided an isolated nucleic acid sequence from BP1155 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP1155 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP1155 bacteriophage of theinvention.

BP1155 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around0.5.

In still another aspect, the invention is related to BP1168bacteriophage, or any variant thereof. BP1168 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainECOR59. BP1168, or any variant thereof, is specific and has lyticactivity against K12/DH5, ECOR13, ECOR15, ECOR28, ECOR35, ECOR46,ECOR50, ECOR54, ECOR55, ECOR59, ECOR71 and/or ECOR72 strains. BP1168comprises a genome comprising a sequence as set forth in SEQ ID NO: 11or having at least 80% identity, more preferably at least 85% identity,and still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO: 11. It is also provided an isolated nucleic acidsequence from BP1168 bacteriophage, or variant thereof. The inventionalso encompasses isolated polypeptides encoded by BP1168 bacteriophage,or variant thereof, or encoded by an isolated nucleic acid sequence fromBP1168 bacteriophage of the invention.

BP1168 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around0.8.

In another aspect, the invention is related to BP1176 bacteriophage, orany variant thereof. BP1176 bacteriophage, or any variant thereof, canbe produced or expanded in e.g., E. coli strain ECOR60. BP1176, or anyvariant thereof, is specific and has lytic activity against K12/DH5,ECOR2, ECOR4, ECOR13, ECOR15, ECOR24, ECOR28, ECOR35, ECOR55, ECOR56,ECOR59 and/or ECOR60 strains. BP1176 comprises a genome comprising asequence as set forth in SEQ ID NO: 12 or having at least 80% identity,more preferably at least 85% identity, and still more preferably 90%,92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 12. It isalso provided an isolated nucleic acid sequence from BP1176bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1176 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1176bacteriophage of the invention.

BP1176 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around4.

In still another aspect, the invention is related to BP1197bacteriophage, or any variant thereof. BP1197 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainK12/DH5. BP1197, or any variant thereof, is specific and has lyticactivity against K12/DH5, ECOR2, ECOR13, ECOR15, ECOR28, ECOR46, ECOR48,ECOR50, ECOR54, ECOR59, ECOR60, ECOR71 and/or ECOR72 strains. BP1197comprises a genome comprising a sequence as set forth in SEQ ID NO: 13or having at least 80% identity, more preferably at least 85% identity,and still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO: 13. It is also provided an isolated nucleic acidsequence from BP1197 bacteriophage, or variant thereof. The inventionalso encompasses isolated polypeptides encoded by BP1197 bacteriophage,or variant thereof, or encoded by an isolated nucleic acid sequence fromBP1197 bacteriophage of the invention.

BP1197 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around1.

In an aspect, the invention is related to BP1226 bacteriophage, or anyvariant thereof. BP1226 bacteriophage, or any variant thereof, can beproduced or expanded in e.g., E. coli strain ECOR59. BP1226, or anyvariant thereof, is specific and has lytic activity against K12/DH5,ECOR2, ECOR13, ECOR28, ECOR48, ECOR59, ECOR60 and/or ECOR72 strains.BP1226 comprises a genome comprising a sequence as set forth in SEQ IDNO: 14 or having at least 80% identity, more preferably at least 85%identity, and still more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or99% identity to SEQ ID NO: 14. It is also provided an isolated nucleicacid sequence from BP1226 bacteriophage, or variant thereof. Theinvention also encompasses isolated polypeptides encoded by BP1226bacteriophage, or variant thereof, or encoded by an isolated nucleicacid sequence from BP1226 bacteriophage of the invention.

BP1226 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around5.

In another particular embodiment, the invention is related to BP1229bacteriophage, or any variant thereof. BP1229 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., E. coli strainK12/DH5. BP1229, or any variant thereof, is specific and has lyticactivity against K12/DH5, ECOR2, ECOR13, ECOR15, ECOR28, ECOR46, ECOR48,ECOR50, ECOR54, ECOR55, ECOR59, and/or ECOR72 strains. BP1229 comprisesa genome comprising a sequence as set forth in SEQ ID NO: 15 or havingat least 80% identity, more preferably at least 85% identity, and stillmore preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 15. It is also provided an isolated nucleic acid sequence fromBP1229 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1229 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1229bacteriophage of the invention.

BP1229 bacteriophage of the invention is also characterized by a PLEbelow 15, more preferably below 10 and still more preferably of around6.

Nucleic Acids and Polypeptides

The invention also relates to a nucleic acid contained in abacteriophage of the invention, or any fragment of such a nucleic acid.The term fragment designates, more preferably, a fragment containing (orconsisting of) an open reading frame. The nucleic acid may be DNA orRNA, single- or double-stranded.

The nucleic acid can be isolated from the deposited bacteriophages, orproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning), enzymatic or chemical synthesis,or combinations thereof, according to general techniques known per se inthe art. Also included are homologous sequences and fragments thereofincluding, but not limited to, natural allelic variants and modifiednucleic acid sequences in which nucleotides have been inserted, deleted,substituted, and/or inverted.

In a particular embodiment, the invention relates to a nucleic acidcomprising a sequence selected from anyone of SEQ ID NOs: 1-15, or asequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to anyone of SEQ ID NOs: 1-15.

In another particular embodiment, the invention relates to a nucleicacid comprising the sequence of a fragment of a sequence selected fromanyone of SEQ ID NOs: 1-15, or a fragment of a sequence having at least90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toanyone of SEQ ID NOs: 1-15, said fragment comprising an open readingframe or a regulatory element such as a promoter.

In a particular embodiment, the invention relates to a nucleic acidhaving or consisting of a sequence selected from anyone of SEQ ID NOs:1-15, or a sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to anyone of SEQ ID NOs: 1-15.

The nucleic acid of the invention can be in free form, or cloned in avector.

In a further aspect, the invention also relates to an isolatedpolypeptide encoded by a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. Thepolypeptides may be produced by techniques known per se in the art suchas synthesis, recombinant technology, or combinations thereof. Thepolypeptides may be isolated or purified, and used as antibacterialagents or as reagents for in vitro analyses.

Compositions of the Invention:

One aspect of the invention relates to compositions comprising at leastone bacteriophage as described above, more preferably at least 2 or moreand, optionally, a pharmaceutically or veterinary acceptable excipient.As described, the bacteriophages of the invention have very potent lyticactivity against E. coli strains. Combinations of these bacteriophagesmay be produced to expand the host spectrum and produce highly effectiveantibacterial compositions.

More particularly, the invention relates to an antibacterial compositioncomprising at least two bacteriophages having lytic activity against anE. coli strain, said at least two bacteriophages being selected from thebacteriophages having a genome comprising a nucleotide sequence ofanyone of SEQ ID NOs: 1 to 15 or a sequence having at least 90% identitythereto, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identity thereto.

In a preferred embodiment, the compositions of the invention comprise atleast three, even more preferably at least four distinct bacteriophagesselected from the bacteriophages having a genome comprising a nucleotidesequence of anyone of SEQ ID NOs: 1 to 15 or a sequence having at least90% identity thereto, preferably at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity thereto. Compositions of the invention maycomprise at least 5, 6, 7, 8, 9, 10, 11, 11, 12, 13, 14 or all of the 15distinct bacteriophages as disclosed above.

One aspect of the invention is related to a composition at least onebacteriophage selected from BP539, BP700, BP753, BP814, BP953, BP954,BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, and/orBP1229, and variants thereof.

The invention also concerns a composition comprising at least twodistinct bacteriophages selected from BP539, BP700, BP753, BP814, BP953,BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226,and/or BP1229, and variants thereof.

Preferably, a composition of the invention comprises at least threedistinct bacteriophages, more preferably at least four distinctbacteriophages, still more preferably at least five distinctbacteriophages, still more preferably at least six distinctbacteriophages, more preferably at least seven distinct bacteriophages,more preferably at least eight distinct bacteriophages, still morepreferably at least nine distinct bacteriophages and still morepreferably at least ten distinct bacteriophages selected from BP539,BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155,BP1168, BP1176, BP1197, BP1226, and/or BP1229, and variants thereof.

In a particular embodiment, a composition of the invention comprisesBP539 in combination with at least one further bacteriophage selectedfrom BP700, BP753, BP814, BP1151, BP1176, and BP1168.

In another particular embodiment, a composition of the inventioncomprises BP1002 in combination with at least one further bacteriophageselected from BP1151, BP1155, BP1168, BP1176 and BP1197.

In another particular embodiment, the composition comprises BP1155 incombination with at least one further bacteriophage selected fromBP1168, BP1197, BP1226, BP1229 and BP1176.

In another preferred embodiment, the composition comprises BP1151 incombination with at least one further bacteriophage selected fromBP1176, BP953, BP970, BP700 and BP1002.

In another preferred embodiment, the composition comprises BP953 and/orBP1168 and/or BP1176, optionally in further combination with at leastone further bacteriophage of the invention.

The invention particularly relates to a composition comprising acombination of bacteriophages BP953+BP1168. Such a composition couldkill 100% tested hemorrhagic E. coli strains and nearly 70% of the 25 E.coli bacteria of Table 2 (see Example 3.1).

The invention also relates to a composition comprising a combination ofbacteriophages BP953+BP1168+BP1229. Such a composition can kill E. colibacteria type 0157, 0144 and 0104, including hemorrhagic strains (seeExample 3.2).

The invention also relates to a composition comprising a combination ofbacteriophages BP953+BP1151+BP1155+BP1176. Such a composition could kill80% of all tested E. coli bacteria isolated from hospitals (see Example3.3).

The invention also relates to a composition comprising a combination ofbacteriophages BP700+BP953+BP970+BP1002+BP1176. Such a composition couldkill 100% of tested ST131-type E. coli bacteria, at least 80% of testedBLSE E. coli bacteria and at least 93% of tested BMR-type E. colibacteria (see Example 3.4).

The invention also relates to a composition comprising a combination ofbacteriophages BP1002+BP1151+BP1155+BP1168+BP1176. Such a compositioncan kill 100% of tested meningitis-causing E. coli bacteria (see Example3.5).

The invention also relates to a composition comprising a combination ofbacteriophages BP539+BP700+BP753+BP814+BP1151+BP1176+BP1168. Such acomposition could kill nearly 96% of the 24 E. coli bacteria of the ECORcollection of Table 2 (see Example 3.6).

The invention also relates to a composition comprising a combination ofall of the bacteriophages BP539, BP700, BP753, BP814, BP953, BP954,BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, and/orBP1229, or variants thereof. Such a composition could kill 100% of the25 E. coli bacteria of Table 2 (see Example 3.7).

Specific examples of compositions of the invention comprise:

-   -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 5 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 11 or a sequence having at        least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 5 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 11 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 15 or a sequence        having at least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 5 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 9 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 10 or a sequence        having at least 90% identity thereto, and a bacteriophage having        a genome comprising a nucleotide sequence of SEQ ID NO: 12 or a        sequence having at least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 2 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 5 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 7 or a sequence        having at least 90% identity thereto, and a bacteriophage having        a genome comprising a nucleotide sequence of SEQ ID NO: 8 or a        sequence having at least 90% identity thereto, and a        bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 12 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 8 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 9 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 10 or a sequence        having at least 90% identity thereto, and a bacteriophage having        a genome comprising a nucleotide sequence of SEQ ID NO: 11 or a        sequence having at least 90% identity thereto, and a        bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 12 or a sequence having at least 90% identity        thereto; or    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 1 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 2 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 3 or a sequence        having at least 90% identity thereto, and a bacteriophage having        a genome comprising a nucleotide sequence of SEQ ID NO: 4 or a        sequence having at least 90% identity thereto, and a        bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 9 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 12 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 11 or a sequence        having at least 90% identity thereto.

A specific embodiment of the invention relates to a compositioncomprising:

-   -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 1 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 2 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 3 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 4 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 5 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 6 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 7 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 8 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 9 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 10 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 11 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 12 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 13 or a sequence having at least 90%        identity thereto;    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 14 or a sequence having at least 90%        identity thereto; and    -   a bacteriophages having a genome comprising a nucleotide        sequence of SEQ ID NO: 15 or a sequence having at least 90%        identity thereto.

The compositions of the invention exert lytic activity towards bacterialpathogen E. coli. The compositions of the invention may further compriseadditional antibacterial agents, particularly other bacteriophageshaving distinct host specificity.

Preferred compositions of the invention are lytic againstantibiotic-resistant E. coli strains.

Further preferred compositions of the invention are lytic against morethat 90% of all bacterial strains of the EcoR collection, a referencecollection of E. coli strains found in nature.

The antibacterial compositions of the invention may be in various forms,such as liquid, semi-liquid, solid or lyophilized formulations.

It is desired in an aspect of the invention that the compositioncomprises between 10^(e2) and 10^(e12) PFU of at least one bacteriophageof the invention, preferably between 10^(e5) and 10^(e10) PFU. When theantibacterial composition comprises several bacteriophages as definedabove, it is preferred that the composition comprises between 10^(e2)and 10^(e12) PFU of each present bacteriophage of the invention.

The compositions of the invention may comprise any effective amount ofthe selected bacteriophage(s). Preferably, they comprise between 10^(e2)and 10^(e12) PFU of each of said bacteriophages, preferably between10^(e5) and 10^(e10) PFU. The relative amounts of each type ofbacteriophage in a composition of the invention may be adjusted by askilled artisan. Typically, when the antibacterial composition comprisesseveral (n) distinct bacteriophages as defined above, the total relativeamount % A of each bacteriophage in the composition is more preferably %A=(100/n₁)×V, wherein n₁ represents the number of distinct types ofbacteriophages and V is a variability factor comprised between 0.2 and5. Most preferably, V is comprised between 0.3 and 3, even morepreferably between 0.5 and 2, generally between 0.8 and 1.5. In atypical embodiment, when the antibacterial composition comprises severalbacteriophages as defined above, it is preferred that the compositioncomprises between 10^(e2) and 10^(e12) PFU of each present bacteriophageof the invention. Preferably, each type of bacteriophage is present in acomposition of the invention in approximately equal relative amounts.

The compositions of the invention preferably comprise a suitable diluentor carrier, such as a pharmaceutically or veterinary acceptableexcipient or carrier. Compositions according to the present inventionmay include any excipient or carrier, such as thickeners, diluents,buffers, preservatives, surface active agents and the like, in additionto the bacteriophage(s) of choice. Such includes physiologicallyacceptable solutions or vehicles that are harmless or do not cause anysignificant specific or non-specific immune reaction to an organism ordo not abrogate the biological activity of the bacteriophage. For liquidformulation, saline, sterile water, Ringer's solution, bufferedphysiological saline, albumin infusion solution, dextrose solution,maltodextrin solution, glycerol, ethanol, and mixtures thereof may beused as a pharmaceutically or veterinary acceptable excipient orcarrier. If appropriate, other conventional additives such asthickeners, diluents, buffers, preservatives, surface active agents,antioxidants and bacteriostatic agents may be added. Further, diluents,dispersants, surfactants, binders and lubricants may be additionallyadded to the composition to prepare injectable formulations such asaqueous solutions, suspensions, and emulsions, oral formulations such aspills, capsules, granules, or tablets, or powdered formulations.Formulations for topical administration may include, band aids,dressings, patches, films, ointments, lotions, creams, gels, drops,suppositories, sprays, nebulizer, tampons, sanitary towels, liquids andpowders. Formulations for decontamination or for medical use may alsoinclude aerosols or sprays.

The compositions of the invention may be used in the medical field,including the human or veterinary medical areas, for e.g. the treatmentof an infection in a mammal or for improving a subject's condition.

The compositions may be used to kill E. coli bacteria in an organism,for treating an infection. The composition may also be used forimproving the condition of a mammal by modifying the microbial flora insaid mammal. In particular, the compositions of the invention canspecifically remove E. coli strains on the skin or mucous membranes of amammal, thus modifying its microbial flora and restoring a properbalance.

In a particular embodiment, the invention also relates to a method fortreating an infection in a mammal comprising the administration to saidmammal of a composition or bacteriophage or nucleic acid or polypeptideas defined above.

In a particular embodiment the method comprises administering at leastone, preferably at least two, even more preferably at least threebacteriophages selected from BP539, BP700, BP753, BP814, BP953, BP954,BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, and/orBP1229, or variants thereof.

The invention also relates to the use of a composition, bacteriophage,nucleic acid or polypeptide as described for the manufacture of amedicament for treating an infection in a mammal, or for restoringmicrobial flora in said mammal.

The compositions or agents of the invention may be administered by anyconvenient route, including intravenous, oral, transdermal,subcutaneous, mucosal, intramuscular, intrapulmonary, intranasal,parenteral, rectal, vaginal and topical. In a preferred embodiment, thebacteriophages or compositions are administered by topical route, e.g.,by application on the skin of a subject. The compositions may beadministered directly or indirectly, e.g., via a support. In thisregard, the compositions may, for example, be applied or sprayed to theafflicted area. Compositions of the invention can also be administeredby oral or parenteral routes. The dosage suitable for applying,spraying, or administrating the compositions of the present inventioncan be adjusted by the skilled person depending on a variety of factorsincluding formulation, mode of administration, age, weight, sex,condition, diet of the mammal being treated at the time ofadministration, route of administration and reaction sensitivity. Aphysician having ordinary skills in the art can readily determine andprescribe the effective amount of the composition required.

The dosing can also be adjusted by the skilled person so that a lyticactivity against antibiotic-resistant E. coli strains is obtained. Anefficient dose to obtain a lytic activity in vivo typically includes aconcentration of at least 10^(e2) PFU/ml, preferably from about 10^(e2)to 10^(e12) PFU/ml, depending on the administration route.Administration may be performed only once or, if needed, repeated.

The compositions of the invention may be administered to treat E. coliinfections, typically respiratory tract, urinary tract, burns, wounds,ear, skin and soft tissue, gastrointestinal or post-surgical infections.

As shown in the experimental section, the bacteriophages andcompositions of the invention are able to selectively kill E. colibacteria in vitro or in vivo. The compositions can destroy mixtures ofdifferent E. coli bacteria, even in vivo, even at low dosage.Furthermore, the compositions of the invention are effective in killingbacteria embedded in biofilms, which is particularly important forpathogenic bacteria. Also, the compositions and bacteriophages of theinvention are strictly unable to affect mammalian cells, and are,therefore, specific and devoid of side effects in vivo.

The invention also relates to the use of a composition, bacteriophage,nucleic acid or polypeptide of the invention for decontaminating amaterial. Due to their potent antibacterial effect, and to their abilityto even compromise the integrity of a bacterial biofilm, thecompositions of the invention can be used as decontaminating agent, toeliminate or at least cause a reduction in bacterial numbers on amaterial. Such methods may be applied for the treatment of a variety ofbiological or non-biological surfaces in both medical and non-medicalcontexts, including solid materials or devices such as, for example,contact lenses, surfaces of devices to be implanted into the body,pipes, ducts, laboratory vessels, textiles, etc.

Diagnostic/Predictive Tests of the Invention:

The invention also concerns a method for predicting or determining theefficacy of a bacteriophage therapy in a subject, wherein the methodcomprises a step of determining a lytic activity of one or morebacteriophage selected from BP539, BP700, BP753, BP814, BP953, BP954,BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, and/orBP1229 to an E. coli strain from a sample from said subject, such alytic activity being indicative of an efficient treatment. In apreferred aspect, the method further optionally comprises a step oftreating said subject by one or more bacteriophages having a lyticactivity to an E. coli strain from a sample of said subject.

In another aspect, the invention provides a method for selecting asubject or determining whether a subject is susceptible to benefit froma bacteriophage therapy, wherein the method comprises the step ofdetermining a lytic activity of one or more bacteriophage selected fromBP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155,BP1168, BP1176, BP1197, BP1226, and/or BP1229 to an E. coli strain froma sample of said subject, a lytic activity of one or more bacteriophageof the invention to at least one E. coli strain indicating a respondersubject.

Another object of the invention relates to a method for predicting theresponse of a subject to a bacteriophage therapy, wherein the methodcomprises the step of determining a lytic activity of one or morebacteriophage selected from BP539, BP700, BP753, BP814, BP953, BP954,BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, and/orBP1229 to an E. coli strain from a sample of said subject, a lyticactivity of one or more bacteriophage of the invention to at least oneE. coli strain being indicative of a good response to said therapy.

Further aspects and advantages of the invention will be disclosed in thefollowing experimental section, which is illustrative only.

EXAMPLES Materials and Methods

Phage Isolation and Preparation

MDR E. coli bacteria were used for isolating and enriching each virulentbacteriophage from environmental water. Environmental samples andovernight culture of bacteria in Luria Bertani (LB) were mixed andincubated at 37° C. for 24 h with shaking to enrich specificbacteriophages. At the end of incubation, drops of chloroform were addedto the culture. The culture was spun down at 11,000 g for 5 minutes toremove bacterial cells and debris. The supernatant was subjected to 0.2μm filter to remove the residual bacterial cells. The enriched phagesolution was plated on LB agar medium with E. coli embedded. Plaquesformed on the plates after 24 h incubation at 37° C. Single plaque waspicked out for subsequent phage purification and amplification. Thephage was then stored at 4° C. in a suspension in LB broth orphysiological saline.

The titer of phage in a suspension was estimated by plaque counting(Postic, 1961). 10-fold dilutions of a suspension were delivered on adried lawn of the propagating strain. The plates were read afterovernight incubation. The plaque-counting method also permittedvisualization of plaque morphology.

Host Range Determination.

The host ranges of bacteriophages were determined among a collection of26 E. coli from the ECOR collection. 10⁹ bacterial cells were mixed withmelted agar and this mixture was poured on solid agar to make doublelayer agar plates. After solidification, isolated bacteriophage stocksolutions were spotted on each plate with different bacterium strain.After allowing 20 min for the spots to be absorbed, the plates wereinverted and incubated for 24 h at 37° C. before the degree of lysis wasrecorded.

Electron Microscopy.

Electron micrographs of each phage were taken with a transmissionelectron microscope.

Sequencing, Analysis and Annotation of Phage Genomes.

To isolate phage DNA, phages were propagated as described above. PhageDNA was isolated by extraction with phenol:chloroform:isoamyl alcohol(25:24:1, VAT), ethanol precipitation and resolution in water. Wholegenome sequencing was done and the BLAST algorithm was used to determinethe similarity to described genes in the National Center forBiotechnology Information [NCBI] database. The genomes were scanned forpotential open reading frames (ORFs).

Example 1 Bacteriophage-Host Characteristics and Kinetics

One-step growth experiments were carried out according to the previousdescriptions to determine first the productive lytic time, adsorptionrate, and then the phage burst size. To determine the adsorption ratesamples were taken at different time intervals to analyze the free phageparticles in the solutions. For productive time and phage burst sizedetermination, E. coli bacteria were mixed with phages solutions andphages were allowed to adsorb for 15 min. The mixture was subjected tocentrifugation immediately at 5000 rpm for 10 min to remove free phageparticles. The pellet was resuspended in 5 fresh LB medium and theculture was continuously incubated at 37° C. Samples were taken at 3 minintervals and phage titre was determined. These results permitted tocalculate the number of phages produced per bacteria (burst size), theproductive time and the productive lytic effect (PLE), as shown in table3 below.

TABLE 3 Productive BURST PLE lytic Adsorption SIZE (PFU per time rate(PFU per bacterium Phage (min) (ml-l min-1) bacterium) per min)  539 709.83E−09 9 0.13  700 15 1.04E−08 32 2.13  753 60 3.00E−08 119 1.98  81430 2.30E−08 9 0.30  953 20 2.05E−08 66 3.30  954 20 2.01E−08 66 3.30 970 10 2.24E−08 46 4.60 1002 10 8.40E−09 49 4.90 1151 10 2.10E−08 999.90 1155 15 1.21E−08 7 0.47 1168 10 8.70E−09 8 0.80 1176 60 1.56E−08232 3.87 1197 45 6.60E−09 49 1.09 1226 30 8.87E−09 149 4.97 1229 556.55E−09 332 6.04

These results show that all phages have potent viral production capacityand absorption rates. Most phages have a PLE below 5, which demonstratesa remarkable profile. Phage 539 is particularly effective in thisregard. In addition, the different PLE and adsorption times permit tocreate cocktails with selected variability.

Example 2 Preparation of a Cocktail Composition

The following cocktail compositions are constituted, each comprisingbetween 10⁻⁹ and 10⁻¹¹ pfu of each bacteriophage:

TABLE 4 Cocktail Phages I BP953 + BP1168 II BP953 + BP1168 + BP1229 IIIBP953 + BP1151 + BP1155 + BP1176 IV BP700 + BP953 + BP970 + BP1002 +BP1176 V BP1002 + BP1151 + BP1155 + BP1168 + BP1176 VI BP539 + BP700 +BP753 + BP814 + BP1151 + BP1176 + BP1168

The following additional two cocktail compositions comprising all of thevarious phages are constituted, covering the most important diversity ofE. coli species.

TABLE 5A cocktail composition A: Phage BP539 BP 700 BP 753 BP 814 BP 953BP 954 BP970 BP1002 Titer 2.72^(E+09) 8.00^(E+09) 2.27^(E+08)5.89^(E+07) 4.53^(E+10) 3.00^(E+08) 4.02^(E+08) 9.73^(E+08) Phage BP1151BP1155 BP1168 BP1176 BP1197 BP1226 B1229 Titer 1.56^(E+09) 3.00^(E+10)7.77^(E+09) 1.00^(E+10) 3.91^(E+09) 4.44^(E+06) 9.11^(E+09)

TABLE 5B cocktail composition B: Phage BP539 BP 700 BP 753 BP 814 BP 953BP 954 BP970 BP1002 Titer 8.85^(E+08) 7.89^(E+08) 7.26^(E+07)3.04^(E+08) 9.47^(E+08) 3.89^(E+08) 9.56^(E+07) 2.09^(E+09) Phage BP1151BP1155 BP1168 BP1176 BP1197 BP1226 B1229 Titer 7.35^(E+08) 2.57^(E+09)3.01^(E+09) 1.77^(E+09) 1.03^(E+10) 2.00^(E+09) 1.56^(E+09)

Example 3 Sensitivity of Bacteria to Bacteriophage Cocktails of theInvention

Various strains of bacteria were tested with a bacteriophage cocktail ofthe invention at 2.10⁹ bacteriophages/ml. Different bacterialconcentrations were plated on the bacteriophage cocktail at 2.10⁹bacteriophages/ml and incubated 24 h at 37° C.

Cocktails are tested on distinct E. coli bacteria listed in table 2 aswell as additional E. coli bacteria, including meningitis-causingbacteria from R. Debré collection (37 strains), BLSE (5 strains) andST131 (9 strains) type E. coli bacteria, E. coli bacteria derived fromhospitalized patients (35 strains), and haemorrhagic E. coli bacteria of0157, 0144 and 0104 type (3 strains). The % of bacteria speciessensitive to the cocktails are listed in table 6 below:

Example 3.1 Efficacy of Cocktail I

As shown in the following Table 6 below, cocktail I is able to destroy100% of tested haemorrhagic E. coli bacteria.

TABLE 6 BP953 BP1168 Cocktail I O157:133 + + O144:227 + + O104:H4 + +

Furthermore, cocktail I can also destroy nearly 70% of the 25 E. colibacteria of table 2.

Example 3.2 Efficacy of Cocktail II

As shown in the following Table 7, cocktail II is able to destroy 100%of tested haemorrhagic E. coli bacteria.

TABLE 7 BP953 BP1168 BP1229 Cocktail II O157:133 + + + O144:227 + + +O104:H4 + +

Furthermore, cocktail II can also destroy 76% of the 25 E. coli bacteriaof table 2.

Example 3.3 Efficacy of Cocktail III

As shown in the following Table 8, cocktail III is able to destroy atleast 80% of tested E. coli strains isolated from hospitalized patients.

TABLE 8 BP953 BP1151 B1155 BP1176 Cocktail III NDM-1 + + SH1 + + +SH4 + + SH5 + + + SH7 + +/− + SH9 + + + SH11 SH12 + + SH25 SH76 + + +SH78 + + SH79 SH80 SH81 +/− + SH84 +/− +/− + SH86 + + SH87 + + + SH88+/− + + SH90 + +/− + + SH92 + + SH95 +/− + SH96 + + SH100 + + +/− +SH102 + + + + SH103 + +/− + SH104 + + SH105 + + + SH107 SH112 + +SH113 + + + SH117 +/− + + SH118 + + SH119 SH120 + + + SH123

Example 3.4 Efficacy of Cocktail IV

As shown in the following Table 9, cocktail IV is able to destroy ST131and BLSE-type E. coli strains.

TABLE 9 BP700 BP953 BP970 BP1002 BP1176 Cocktail IV ST131TN03 + + + + + + ST131 +/− + + RD20873 ST131 + + +/− +/− + + RD 5530ST131 XXF +/− +/− + + ST131 XXT + + +/− + + + ST131 6601 + + + + + ST13127144 + + + ST131 28678 +/− + + ST131 30151 + + +/− + + BSE3 + + +BSE4 + +/− + BSE7 + + BSE9 +/− + +/− + + BSE12

Example 3.5 Efficacy of Cocktail V

As shown in the following Table 10, cocktail V was able to destroy 100%meningitis-causing E. coli bacteria from R Debré collection.

TABLE 10 BP1002 BP1151 BP1155 BP1168 BP1176 BP1197 Cocktail V CFT073+/− + + J96 +/− +/− + + 536 + + +/− + + + S5 + + + + + + S11 + + + + + +S12 + + + + + + + S13 + +/− + S15 + +/− + S22 + + + S25/C5 + +/− +S39 + + + + S41 +/− +/− + + + + S43 + +/− + S49 + + + + + S51 + + + + +S55 +/− + + + S63 + + + S69 + +/− + + + + S88 +/− +/− +/− +/− +/− +/− +S97 +/− + +/− +/− + +/− + S102 + + + + + S104 +/− + +/− + +/− + S105+/− + + + S106 +/− + + + + + + S113 + + + + S120 +/− + S123 +/− + +/− +S124 + + + S130 +/− + + S133 +/− + + S138 + +/− + S149 + + + + S176 +/−+/− + S182 + + + + S191 +/− + + + S192 + + + + S242 + + +

Example 3.6 Efficacy of Cocktail VI

Cocktail VI is able to destroy nearly 96% of the 24 E. coli bacteria ofECOR collection as listed in table 2.

Example 3.7 Efficacy of Cocktails A and B

Cocktails A and B are both able to 100% of the 25 E. coli bacterialisted in table 2.

Bacteria were further enumerated and used to the calculation ofresistance rate (number of bacteria after incubation/number of bacteriaplated). Resistance rates with cocktail A comprising the 15 differenttypes of bacteriophages are shown in the following table 11:

TABLE 11 Rate Bacteria (bacteria/ml) ECOR1 >1.00E−02 ECOR24 2.00E−05ECOR60 4.00E−06 S22 1.18E−04 S106 <1.00E−06 S182 2.00E−06 SH5 1.00E−06SH113 1.00E−06 Astrid 9 <1.00E−06 BSE 3 8.50E−05 BSE 7 5.00E−06 0157:1332.63E−04 XXT 1.74E−04

All tested bacteria are sensitive to compositions of the invention.

Example 4 Cocktail Specificity

The cocktail specificity was confirmed by testing on ten bacteriaspecies, including Pseudomonas aeruginosa, Acinebacter baumanii,Enterobacter aerogenes C, Enterobacter asburiae, Enterobacter cloacae,Klebsiella pneumoniae, Proteus mirabilis, Staphylococus aureus,Stenotrophomonas maltophila, Serratia marcescens.

Table 12 summarizes lytic activity observed for each bacteriophage usedindependently or in combination as a cocktail of 15 bacteriophages.

TABLE 12 Bacterium E54 E55 E56 E55 E55 E35 DH5 E56 E56 E59 E59 E60 DH5E59 DH5 Cocktail Phage EC 539 700 753 814 953 954 970 1002 1151 11551168 1176 1197 1226 1229 15 phages E. coli SH 213 +/− +/− +/− +/− + + ++/− + + SH 141 +/− +/− +/− +/− +/− +/− Pseudomonas SH 85 − aeruginosa SH224 − Acinebacter SH 32 − baumanii SH 34 − Enterobacter SH 97 −aerogenes C SH 98 − Enterobacter SH 74 − asburiae Enterobacter SH 111 −cloacae SH 121 − Klebsiella SH 89 − pneumoniae SH 283 − Proteusmirabilis SH 82 − Staphylococus SH 14 − aureus SH 129 − StenotrophomonasSH 286 − maltophila SH 290 − Serratia SH 314 − marcescens

The above table clearly show that no lytic activity on bacteria otherthan E. coli strain occurred. The bacteriophages and cocktail of theinvention are therefore highly specific for E. coli strains.

Example 5 Efficiency of Bacteriophages on E. coli Strain In Vitro

Several strains of the EcoR collection were chosen to represent thegenetic diversity of E. coli and various forms of antibiotic resistance.Strains were either sensitive or resistant to one or severalantibiotics. They were grown individually or in combination with 2 to 8strains. The bacteriophage cocktail was added at a MOI of 1 to 10^(e−6),i.e. at a dilution ratio (bacteria/phage) of 1 to 1 million.

The results are presented in FIG. 1 and in the following Table 13.

TABLE 13 Efficiency of bacteriophage cocktail obtained in vitro on E.coli mixture: at 2.10^(e7) cfu/ml and at various dilutions: MOI MOI MOIMOI MOI MOI Mix: 1 0.1 0.01 0.001 0.0001 0.000001 1 bacterium ++ ++ ++++ + + 2 bacteria ++ ++ + +/− +/− +/− 3 bacteria ++ ++ + + +/− +/− 4bacteria ++ ++ ++ + +/− +/− 5 bacteria ++ ++ ++ + + +/− 6 bacteria ++++ + +/− +/− +/− 7 bacteria ++ ++ ++ + +/− +/− 8 bacteria ++ ++ ++ + +/−The compositions of the invention are able to kill a mixture of 8distinct strains of E. coli bacteria. The cocktail remains efficientagainst 8 strains at a dilution of 1/1000.

Example 6 Efficiency of Bacteriophages on E. coli Strain In Vivo

An isolated SH113 strain, collected on a burned patient in 2011, wasused for the following experiments.

SH113 strain is resistant to ampicillin, ticarcillin, cefalotin,cefotaxim, nalidixic acid, norfloxacin, ofloxacin, ciprofloxacin.

SKH1 mouse (or hairless mouse) was used as mouse model of E. coliinfection.

Modus operandi: (see table 14 below)

-   -   Mice were immunodepressed by 3 IP injections of 1.5 mg of        cyclophosphamide (Cy), every 2 days from the Day −3 before        infection.    -   Mice were burned on skin by 2 μl of liquid yperite at 30 mg/kg.    -   Infection two days after the burn by subcutaneous injection of a        bacterium suspension in burned site.

TABLE 14 −3 −2 −1 0 1 Day 1.5 mg Cy Burn 1.5 mg Cy Infection 1.5 mg CyInjection IP Yperite IP SC 10⁷ cfu IP route PHAGE SC, IV or IP injectionof cocktail (100 μl, i.e. 10⁸ PFU) 6 h post- infection

Cocktail compositions were prepared according to example 2 andcompresses were soaked with bacteriophages cocktail at 10^(e7) phages/mlbefore applying at Day 0.

Various concentrations of E. coli strains were tested with 100 μl ofbacteriophage cocktail. As shown on FIG. 2, all E. coli strains werekilled 6 h post-treatment.

Upon administration of SH113 E. coli strain by sub-cutaneous injectionto SKH1 mice, all mice died in the absence of further treatment. In themice treated by injection of a bacteriophage cocktail as presented intable 9 above, a remarkable survival rate was observed (see FIG. 3):100% survival for SKH1 mice treated subcutaneously or intravenously and65% survival for intraperitoneal route treatment. By comparison, a 80%survival rate was observed for SKH1 mice treated by a double dose ofgentamicin antibiotic at Day 0+6 h and during 7 consecutive days,including 2 injections on Day 1.

A remarkable 100% survival rate was also obtained after subcutaneoustreatment with 1/10, 1/100 and 1/1000 (i.e. 10⁵ PFU per mouse) dilutionsof the cocktail of the invention (see FIG. 4).

Accordingly, the compositions of the invention can treat an infection invivo and can induce a 100% survival rate in infected mice.

REFERENCES

-   Afifi, R. Y., and A. A. El-Hindawi. 2008. Acute necrotizing    fasciitis in Egyptian patients: a case series. Int. J. Surg. 66-14.-   Brzozowski D., and D. C. Ross. 1997. Upper limb Escherichia coli    cellulitis in the immunocompromised. J. Hand Surg. 22678-680-   Clark W A, 1962, Appl Microbiol. Comparison of several methods for    preserving bacteriophages. 1962 September; 10:466-71.-   Corredoira, J. M., J. Ariza, R. Pallares, J. Carratala, P. F.    Viladrich, G. Rufi, R. Verdaguer, and F. Gudiol. 1994. Gram-negative    bacillary cellulitis in patients with hepatic cirrhosis. Eur. J.    Clin. Microbiol. Infect. Dis. 1319-24-   Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Delattre AS,    2012, Learning from bacteriophages—advantages and limitations of    phage and phage-encoded protein applications; 13(8):699-722.-   Fraser, N., B. W. Davies, and J. Cusack. 2006. Neonatal omphalitis:    a review of its serious complications. Acta Paediatr. 95519-522.-   Krebs, V. L., K. M. Koga, E. M. Diniz, M. E. Ceccon, and F. A.    Vaz. 2001. Necrotizing fasciitis in a newborn infant: a case report.    Rev. Hosp. Clin. Fac. Med. Sao Paulo 5659-62.-   Needleman S B, Wunsch C D “A general method applicable to the search    for similarities in the amino acid sequence of two proteins.” 1970    March; 48(3):443-53.-   Rodgers, G. L., J. Mortensen, M. C. Fisher, A. Lo, A. Cresswell,    and S. S. Long. 2000. Predictors of infectious complications after    burn injuries in children. Pediatr. Infect. Dis.; 19(10):990-5.-   Stone R. 2002. Bacteriophage therapy. Stalin's forgotten cure.    Science 298, 728-731 (DOI: 10.1126/science. 298.5594.728)-   Tourmousoglou, C. E., E. C. Yiannakopoulou, V. Kalapothaki, J.    Bramis, and J. St. Papadopoulos. 2008. Surgical-site infection    surveillance in general surgery: a critical issue. J. Chemother.    20(3)312-318.-   Weinbauer MG. Ecology of prokaryotic viruses. FEMS Microbiol Rev    2004; 28:127-81.

The invention claimed is:
 1. A method of treating of an infection in amammal in need thereof, comprising administering to the mammal infectedwith an Escherichia coli (E. coli) strain an antibacterial compositionin an amount effective to treat said E. coli infection, wherein saidcomposition comprises at least two distinct bacteriophages having lyticactivity to said E. coli strain, said at least two distinctbacteriophages being selected from the bacteriophages having a genomecomprising a nucleotide sequence selected from any one of SEQ ID NOs: 1to 15 or a sequence having at least 99% identity thereto.
 2. The methodof claim 1, wherein the composition comprises at least three distinctbacteriophages selected from the bacteriophages having a genomecomprising a nucleotide sequence selected from any one of SEQ ID NOs: 1to 15 or a sequence having at least 99% identity thereto.
 3. The methodof claim 1, wherein the composition comprises a combination of all ofthe bacteriophages BP539, BP700, BP753, BP814, BP953, BP954, BP970,BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and BP1229comprising the nucleotide sequence of SEQ ID NOs: 1 to 15, respectively.4. The method of claim 1, wherein the composition is lytic againstantibiotic-resistant E. coli strains.
 5. The method of claim 1, whereinthe composition is lytic against more than 90% of all bacterial strainsof EcoR collection.
 6. The method of claim 1, wherein the compositionfurther comprises a pharmaceutically acceptable excipient or carrier. 7.The method of claim 1, wherein the composition is a liquid, semi-liquid,solid or lyophilized formulation.
 8. The method of claim 1, wherein thecomposition comprises between 10^(e2) and 10^(e12) PFU of bacteriophage.9. A method for improving the condition of a mammal by modifying themicrobial flora, comprising administering to the mammal an effectiveamount of an antibacterial composition, wherein said compositioncomprises at least two distinct bacteriophages having lytic activity toan Escherichia coli (E. coli) strain, said at least two distinctbacteriophages being selected from the bacteriophages having a genomecomprising a nucleotide sequence selected from any one of SEQ ID NOs: 1to 15 or a sequence having at least 99% identity thereto and saidmicrobial flora comprises said E. coli strain.
 10. A method fordecontaminating a material, comprising exposing the material to anamount of an antibacterial composition effective to decontaminate saidmaterial, wherein said composition comprises at least two distinctbacteriophages having lytic activity to an Escherichia coli (E. coli)strain, said at least two distinct bacteriophages being selected fromthe bacteriophages having a genome comprising a nucleotide sequenceselected from any one of SEQ ID NOs: 1 to 15 or a sequence having atleast 99% identity thereto.
 11. An antibacterial composition comprisingat least two distinct bacteriophages having lytic activity to anEscherichia coli (E. coli) strain and a pharmaceutically acceptableexcipient or carrier, said at least two distinct bacteriophages beingselected from the bacteriophages having a genome comprising a nucleotidesequence selected from any one of SEQ ID NOs: 1 to 15 or a sequencehaving at least 99% identity thereto and said pharmaceuticallyacceptable excipient or carrier comprising a preservative in an amounteffective to preserve the activity of the bacteriophages.
 12. Thecomposition of claim 11, comprising at least three distinctbacteriophages selected from the bacteriophages having a genomecomprising a nucleotide sequence selected from any one of SEQ ID NOs: 1to 15 or a sequence having at least 99% identity thereto.
 13. Thecomposition of claim 11, comprising a combination of all 15 of saidbacteriophages.
 14. The composition of claim 11, which is a liquid,semi-liquid, solid or lyophilized formulation.